This project seeks to elucidate the role of carnitine acetyltransferase (CrAT) as an important regulator of mitochondrial function and glucose tolerance, and the principal mediator of the antidiabetic actions of L-carnitine therapy. Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that plays a key role in the synthesis and efflux of short chain carnitine conjugates, such as acetyl-carnitine. This enzyme is most abundant in carnitine-rich tissues such as skeletal muscle and heart, but its precise metabolic function remains largely unexplored. CrAT activity is mainly regulated by availability of L-carnitine, a conditionally essential nutrient that is best known for its obligatory role in permitting mitochondrial uptake and oxidation of long chain fatty acids. In addition to its requisite role in fat oxidation, carnitine also permits the intramitochondrial formation of acylcarnitine conjugates, thereby facilitating mitochondrial efflux of excess carbon fuels. Recent studies by our laboratory suggest that carnitine insufficiency caused by aging and/or overnutrition impairs fuel metabolism and insulin action by compromising CrAT activity. Remarkably, dietary carnitine supplementation improved metabolic outcomes in these models in association with robust increases in plasma and urinary acetyl-carnitine levels. The physiological relevance of acetyl-carnitine production and efflux is poorly understood and surprisingly understudied. We seek to understand the specific role of CrAT as a carnitine effector that defends metabolic homeostasis. We will address two central hypotheses: 1) CrAT plays a key role in regulating mitochondrial substrate switching between glucose and fatty acid fuels, and 2) mitochondrial efflux of CrAT-derived acylcarnitines affords protection against muscle insulin resistance and oxidative stress caused by chronic overnutrition. These hypotheses will be tested using gain- and loss-of-function genetic engineering approaches in primary human skeletal myocytes as well as knockout mouse models. Primary outcome measures will include indirect calorimetry, multiple measures of insulin action and metabolic flux, along with state-of-the-art mass spectrometry-based metabolic profiling.